Structure-activity relationships of chloride-sensitive fluorescent indicators for biological application

ANALYTICAL

169, 142-150

BIOCHEMISTRY

Structure-Activity

RETO KRAPF,’

(1988)

Relationships of Chloride-Sensitive Indicators for Biological Application

NICHOLAS

P.

ILLSLEY,

HSIEN

C. TSENG,

Fluorescent

AND A. S. VERKMAN

Cardiovascular Research Institute, University of California, San Francisco, California 94143-0532 Received

August

27, 1987

The application of the quinoline derivative 6-methoxy-N-( 3-sulfopropyl)quinolinium (SPQ) to the measurement of membrane transport of chloride in biological systems was reported recently (N. P. Illsley and A. S. Verkman (1987) Biochemistry 26, 12 I5- I2 19). To understand the structure-activity relationships of compounds with chloride-sensitive fluorescence properties, 19 structural analogs of SPQ having a single quaternized nitrogen heteroatom were synthesized and characterized. The effect of variations in ring structure, length of sulfoalkyl chain, position of ring substituent, and nature of ring substituent were examined. For each compound, the water solubility, octanokwater partition coefficient. absorbance and fluorescence spectra, fluorescence lifetime, and Stern-Volmer constants (K,) for quenching by a series of anions were measured. All compounds were quenched by chloride, bromide, iodide, and thiocyanate, but not by cations, sulfate, phosphate. nitrate. or by pH (5-8): several compounds were quenched slightly by bicarbonate (K, = 8-12 Mm ‘). High chloride sensitivity (k; > 50 M ‘) required the presence ofa quinoline backbone substituted with electron-donating groups such as methyl and methoxy. but did not depend on length of the sulfoalkyl chain or on the position of ring substituents (positions 2-7). All compounds with high chloride sensitivity had fluorescence excitation spectra in the ultraviolet (excitation maximum ~350 nm) and fluorescence lifetimes > 15 ns. These results establish a set of guidelines for synthesis of chloride-sensitive fluorescent indicators tailored for specific biological applications. G 1988 Academic PESS. IIIC KEY WORDS: fluorescent dyes: chloride: quinoline; absorbance: fluorescence; membrane transport.

Membrane transport of chloride has an important role in cellular regulatory, absorptive, and secretory processes. Studies of chloride movement in biological systems have been complicated by technical limitations of current methods to measure chloride concentration. Patch-clamp techniques apply only to conductive transport; microelectrodes have limited specificity, sensitivity, and response rate; and 36C1 used in tracer studies has low specific activity. Based on the success of pH and calcium measurements using intracellular fluorescent indicators, the measurement of cell chloride with fluorescent dyes would be an ideal non’ To whom correspondence should be addressed. 0003-2697/88

and

$3.00

Copyright Q 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

reprint

invasive method. Wolfbeis and Urban0 (1) have reported the synthesis of quinoline and acridine compounds with halide-sensitive fluorescence properties. Recently, we reported that one of these substances, 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ)’ can be used to study chloride transport in isolated membrane vesicles (2-6). Chloride quenching of SPQ fluorescence (excitation 350 nm, emission 450 nm) occurs by a collisional mechanism (2). Compared to “Cl methods, the SPQ method has much improved sensitivity and time resolution in the study of membrane vesicle chloride trans* Abbreviations pyl)quinolinium; 4-(2-hydroxyethyl)-

requests

142

SPQ. 6-methoxy-N-(3-sulfopro&, Stern-Volmer constant; Hepes, I -piperazineethanesulfonic acid.

used:

STRUCTURE-ACTIVITY

OF CHLORIDE-SENSITIVE

port. In addition, SPQ has been loaded into cells of an intact epithelium (isolated rabbit proximal convoluted tubule) to estimate cell chloride activity (7). An ideal chloride-sensitive fluorophore for use in biological systems should have the following characteristics: (1) high sensitivity and selectivity for chloride; (2) good cell loading and trapping; (3) high quantum yield and molar extinction, and (4) low cell toxicity. It would be desirable to develop compounds with emission peak wavelengths ~500 nm to minimize interference by cell autofluorescence. In addition, it would be advantageous to develop compounds with chloride-dependent fluorescence excitation or emission spectra so that absolute cell chloride activity can be determined from spectral shape, without knowledge of absolute cell indicator concentration. There is little known about the mechanisms and structure-function relationships for quenching of fluorophores by chloride. Because there are no well-established photochemical principles to predict chloride sensitivity, it was necessary to examine the properties of a series of compounds to establish a set of empirical relationships. SPQ was taken as the reference compound in investigations designed to examine the effects of: (1) changing the heterocyclic backbone: (2) varying the length of the sulfoalkyl group: (3) varying the substituent position on the ring molecule, and (4) altering ring substituents. We correlated compound structure with physicochemical properties (water solubility, octanol:water partition coefficient), optical characteristics (molar extinction, fluorescence lifetimes, excitation and emission spectra), and the Stern-Volmer constants for quenching by anions. This structure-activity information is essential for the development of “tailored” fluorescent indicators for application to biological systems, including: ( 1) indicators capable of being trapped intracellularly; (2) indicators with chloride-sensitive and chloride-insensitive fluorescence peaks.

FLUORESCENT

and (3) impermeable cell injection.

INDICATORS

143

chloride indicators for

METHODS

Synthesis procedures. The nitrogen heteroatom of a series of heterocyclic compounds were quaternized with a 3-sulfopropyl or 4sulfobutyl group by reaction with 1,3-propane sultone or 1,4-butane sultone based on the procedure described by Wolfbeis and Urban0 ( 1). The reactants were mixed in a 1:2 heterocyclic compound:sultone mole ratio and heated in an oil bath under exclusion of moisture (flowing Nz) until the heterocyclic reactant disappeared, as determined by thinlayer chromatography. The product compounds either were dissolved and recrystallized from water/ethanol ( 1: 1, v/v) or were extracted in water/chloroform (1:4, v/v) and lyophilized. The latter procedure gave effective product purification because the products were polar and water soluble while the reactants were nonpolar. Table 1 lists reaction time and temperature and the extraction procedures used. All reactants were purchased from Aldrich Chemical Co. (Milwaukee, WI). N-( 3-sulfopropyl)acridinium was purchased from Molecular Probes (Eugene, OR) and N-( 3-sulfopropyl)pyridinium was purchased from Aldrich. Product purity was confirmed by reversephase thin-layer chromatography using a methanol:chloroform (1:35, v/v) solvent system. Product structure was confirmed for all compounds by infrared spectroscopy using a KBr pellet and for compounds 1,4,9, and 15 by nuclear magnetic resonance using a 1% solution in D20. Water solubility was determined from the optical absorbance of the supernatant of saturated dye solutions at 23°C. 0ctanol:water partition coefficients were obtained by a double extraction procedure: 200 ~1 of a 1 mM aqueous dye solution was added to a mixture of 2.5 ml of water and 1 ml of octanol. After vigorous vortexing, 0.5 ml of the octanol supernatant was removed and added to 2.5 ml of water. The fluorescence of

144

KRAPF

ET AI

the aqueous phase was measured and compared to that measured for aqueous solutions of known dye concentrations. Fluorescence

measurements were performed at dye concentrations c 0.1 mM to avoid inner filter effects.

TABLE SYNTHESIS

Reaction temperature Compound

No.

Ring structure R;-(3-Sulfopropyl) quinolinium N-(4-Sulfobutyl) quinolinium N-(3-Sulfopropyl) isoquinolintum K(4Sulfobutyl) tsoquinohnium IV-(3-Sulfopropyl) actidinium ‘v-(3-Sulfopropyl) phenantridinium N-(3-Sulfopropyl) -(5.6-henzyl)quinolininm A’-(3-Sulfopropyl) -(7.8-benzyl)quinolinium Ring substituents 6-Methoxy-n;-(3-sulfopropyl)quinolinium 6-Methoxy-N-(4-sulfobutyl)quinolinium 6-( Methyl-IV-(3.sulfopropyl)quinolinium 2.Methy1.6-Methoxy-A (3-sulfopropyl)quinolinium) 3.Bromo-A’-(3-sulfopropyl)quinolinium 4.Chloro-N-(3-sulfopropyl)quinolinium Ring position 2-Methyl-N-(2-sulfopropyl)quinolinium 4-Methyl-N-(3-sulfopropyl)quinolinium 7-Methyl-A’-(3-sulfopropyljquinolinium 8-Methyl-N-(3.sulfopropyl)quinolinium 2,6-Dimethyl-A’-(3-sulfopropyl)quinolintum 2.6-Dimethyl-N-(4-sulfobutyl)quinolinium a W, water

extraction:

(“Cl

AND

SOLUBILITY

Reaction time (mitt)

I PROPERTIES

Extraction procedure”

Description compound

of

Solubihty tn Hz0 CM)

Octanol:H>O partition coefficient (x10-J)

I

100

30

W

White

crystals

0.90

0.30

2

100

60

W

Whtte

ctystals

I.20

0.2x

3

60

I5

W

~‘rllou

crystals

I OCI

0.62

4

100

30

W

Yellow

crystals

1.73

0. I I

I’ellow

crystals

0.007

4.96

5 6

200

30

W

Whtte

crystals

0.003

4.75

7

150

I80

W

Brown

crystals

0.004

7.46

8

200

30

W

Whne

crystals

II 01

6.04

9

IO0

30

RE

Whtte

crystals

0.90

I .94

10

100

I5

RE

Whne

crystals

0.43

0.71

II

I50

I80

W

0.08

0.92

12

150

I80

W

Light yellow 011 Black crystals

0.54

I .05

13

I00

I20

w

0.18

0.97

14

loo

I20

W

White-yellow crystals Light yellow crystals

n.14

I .07

15

100

30

W

Brown

0.51

0.4 I

16

I20

30

W

0.1 I

0.52

17

150

I20

W

0.16

0.88

18

200

I20

W

Ltght brown oil Light yellow crystals Yellow crystals

0. I7

1.36

19

150

I80

W

Purple

crystals

0.28

1.07

20

I50

60

W

Purple

crystals

0.25

0.56

RE, recrystallization

(see text)

oil

STRUCTURE-ACTIVITY

OF CHLORIDE-SENSITIVE

Characterization of absorbance and jluorescence properties. Absorbance spectra were performed using a photodiode array spectrometer on samples dissolved in 100 mM Na2S04, 5 mM Hepes/Tris, pH 7.4. Sample concentrations were adjusted to give a maximum optical density of 0.3-0.4 at l-cm pathlength. Molar extinction coefficients for the compounds were calculated using the Beer-Lambert Law from optical densities measured at the peak absorbance. Measurements of fluorescence spectra, intensities, and lifetimes were carried out on an SLM 4800 subnanosecond fluorometer (SLM Instruments, Urbana, IL) equipped with a thermostated cuvette holder and interfaced to an IBM PC/XT computer. Samples were measured in acrylic cuvettes, maintained at 23”C, and mixed continuously by a magnetic stirring bar. Excitation and emission spectra were recorded at dye concentrations of 0.1 mM in distilled water. Sample fluorescence intensities (0.1 KIM) were measured relative to SPQ (0.1 mM) in 5 mM Hepes/Tris, pH 7.4, using the excitation and emission maxima for each compound. Fluorescence lifetimes were determined by the phase-modulation technique (89) at a frequency of 18 MHz. Fluorescence was excited at the peak excitation wavelength and detected using Schott KV-408 cut-on filters. Sample lifetimes were measured at a concentration of 0.3 KtM in either water or 100 mM Na2S04, 5 mM Hepes/Tris, pH 7.4, using the reference dimethyl- 1,4-bis(4-methyl-%phenyloxazol-2-yl)benzene in ethanol [lifetime 1.45 ns (lo)]. Fluorescence intensities of sample and reference were matched to within 5% for all lifetime determinations. A single lifetime component was detected for each compound as judged by agreement between phase and modulation lifetimes measured at 18 MHz, suggesting the presence of a single fluorescent species. Fluorescence quenching studies. Compounds being tested as quenchers were added to 0.25 mM fluorophore in 100 mM Na$O,, 5 mM Hepes/Tris, pH 7.4, from a 0.5 M or 1

FLUORESCENT

INDICATORS

145

M stock of the sodium

salt of the quencher. Fluorescence intensities measured after addition of quencher were corrected for dilution and used to calculate the Stern-Volmer quench constant according to the equation FoIF = 1 + KJQ], where FO and Fare the fluorescence intensities in the absence and presence of the quencher, [Q] is the concentration of the quencher, and & is the Stern-Volmer constant. Z$ was obtained by a linear regression using Eq. [ 11. RESULTS

AND

DISCUSSION

Synthesis and PhJaicochemical

Properties

Figure 1 shows the molecular structures of the quinoline, isoquinoline, acridine, and phenanthridine rings. The genera1 quaternization reaction of the nitrogen heteroatom is shown. The product is a zwitterionic inner salt with an N-substituted sulfoalkyl group. Table 1 gives the reaction conditions for each compound. The octanol:water partition coefficient is an estimate of lipid permeability and correlates inversely with the water solubility as illustrated in Table 1. The sulfobutyl com-

7 8

i4 2

6c3J 5

4

3

QUINOLINE

ISOPUINOLINE

ACRIDINE

PHENANTHRIDINE

Ho 0 s=o + c ‘0

Mso,o

lN\

-

AN\

FIG. I. Structures of compounds with a single nitrogen heteroatom. The numbering system of quinoline is shown. Quatemization of the nitrogen heteroatom with propane s&one to form the zwitterionic inner salt is shown.

146

KRAPF

pounds were more soluble in water and less lipid permeable (compound pairs 1 and 2, 3 and 4, 9 and 10, 19 and 20). For the quinoline derivatives, the most chloride-sensitive dyes (see below), substitution with methoxy and methyl groups (compounds 9, 11, and 15-M) increased the octanol:water partition coefficient. The 3-ringed structures (compounds 5-8) had the lowest water solubilities and highest octanol:water partition coefficients. Data from previous studies allow correlation of the octanol-water partition coefficient with apparent membrane permeability for SPQ (3,4,7). SPQ (compound 9) crosses cell membranes with an apparent half-time of >8 h at 0°C 30-90 min at 23°C and lo-30 min at 37°C. Rapid cell loading of SPQ is possible using high dye concentrations (5-20 IrIM), giving suitable intracellular or intravesicular concentrations in a few minutes. Once loaded, dye leakage occurs with the same half time as for loading, a time short compared to the time required for most experimental protocols. The measured octanol:water partition coefficient of the synthesized compounds can be used as an approximate measure of the half-time for compound movement across cell and vesicle membranes relative to that of SPQ. Structural-Optical

Corrdations

Table 2 lists the molar extinction coefficients, fluorescence lifetimes, the peak excitation and emission wavelengths, and the fluorescence intensities of each substance relative to SPQ. In Fig. 2, the excitation and emission spectra for compounds 7, 10, and 16 are shown as representative examples. In Table 3, the Stern-Volmer constants are compiled for Cl-, I-, Bra, SCN-, SO:-, HCOi, NO;, HPOi-, and citrate. Figure 3 shows the Stern-Volmer plots for chloride of substances 3,8,9, and 11. With reference to compound 9 (SPQ), we first examined the effect of changes in the ring structure on the fluorescence properties and the sensitivity to quenching of fluores-

ET AL.

cence by anions. As shown in Table 3, the chloride sensitivity, as measured by the Stern-Volmer quench constant of compounds with an unsubstituted quinoline ring structure (compounds 1 and 2), is superior to isoquinoline (compounds 3 and 4) and phenantridine (compound 6) compounds. Chloride sensitivity is greatly reduced with benzoquinoline (compounds 7 and 8) and acridine (compound 5) derivatives. The fact that these ring structures preserve iodide and bromide sensitivity suggests that a quinoline backbone has a specific role in chloride sensitivity of the compounds. As expected, the excitation and emission peak wavelengths are red-shifted with increasing numbers of rings (Table 2. acridine derivative and benzoquinolines, compounds 5,7, and 8). However, the potential advantage of a spectral response at visible wavelengths is offset by the decrease in chloride sensitivity. The equivalent single ring structure, N-(3-sulfopropyl)pyridinium, is nonfluorescent. The influence of the length of the sulfoalkyl group on fluorescence properties and anion sensitivities was next studied. The sulfopropyl and sulfobutyl derivatives of quinoline (compounds 1 and 2) 6-methoxyquinoline (compounds 9 and lo), 2,6-dimethylquinoline (compounds 19 and 20), and isoquinoline (compounds 3 and 4) were compared. As shown in Table 3, elongation of the sulfoalkyl group by one carbon atom does not alter anion sensitivity of the quinoline and 2,6-dimethylquinoline derivatives significantly, Interestingly, however, elongation of the sulfoalkyl group decreases the chloride sensitivity of the derivative most sensitive to chloride, compound 9. In addition, the length of the sulfoalkyl chain uniformly had little effect on the absorbance and fluorescence properties of the synthesized compounds (Table 2). To examine whether substitution at position 6 of the quinoline ring is important for chloride sensitivity. we studied the fluorescence characteristics of monomethyl derivatives of N-(sulfopropyl)quinolinium substi-

STRUCTURE-ACTIVITY

OF

CHLORIDE-SENSITIVE TABLE OPTICAL

FLUORESCENT

INDICATORS

147

2

PROPERTIES Fluorescence

Compound”

Peak absorbance (nm)

Ring structure 1 2 3 4 5 6 7 8

lifetimes Molar extinction coefficient (M-l)

(nsec)

Buffer

Hz0

Peak excitation/ peak emission (nm)

Relative intensity

318 320 340 340 360 330 370 370

6,100 2,300 2.900 3.000 13.300 3,100 4.500 2.900

7.7 Il.4 12.5 12.3 23.3 7.8 7.8 5.8

13.1 15.0 13.1 13.7 31.4 8.2 8.6 6.5

328/400 3X/400 336/380 3361376 400/490 3681406 3721434 3701434

0.75 0.56 1.62 1.64 I.61 0.61 I .64 I .09

12 13 14

318 318 320 315 323 320

4,700 9,400 10.500 4.200 4.400 5.800

22.7 22.2 14.7 16.9 7.9 ‘7.6

‘5.3 24.5 19.9 70.9 8.6 2.5

3501442 3501440 355/410 3461436 3301426 3321406

1.00 0.98 0.77 0.86 0.16 0.13

Ring position 15 16 17 18 19 20

320 315 322 320 322 322

9.050 7.300 4.300 6,200 5.800 6,300

12.0 12.4 16.0 28.7 17.5 17.0

16.0 16.9 19.5 39.3 21.8 20.0

3141428 3451400 3341406 3361484 320/406 3301406

Ring substituents 9 10 11

a Compounds

are given

by number;

see Table

1 for chemical

tuted at positions 2 (compound 15) 4 (compound 16) 6 (compound ll), 7 (compound 17) and 8 (compound 18), and a dimethylsubstituted derivative (methylation on ring positions 2 and 6, compounds 19 and 20). As shown in Tables 2 and 3, the anion sensitivities and the fluorescence properties of these compounds are quite similar. A notable exception is substance 18, the 8-methyl derivitive, which had a very long fluorescence lifetime, a red-shifted emission spectrum, and decreased chloride sensitivity. Substitution at the 8-position appeared to give unique optical and quenching properties because two other compounds tested [8-ethoxy-N-( 3-sulfopropyl)quinolinium and 8-acetoxy-N-(3sulfopropyl)quinolinium] had emission

1.63 1.30 0.96 0.82

names

peaks at -500 nm and Stern-Volmer constants for chloride quenching ~20 M-’ (data not shown). A comparison of the fluorescence properties of the methyl derivatives with the unsubstituted quinolines (compounds 1 and 2) shows that ring substitution with methyl or methoxy groups by itself and irrespective of the ring position confers increased chloride sensitivity. Because of the increased chloride sensitivity conferred by methyl and methoxy substitution on the quinoline ring. the 2-methyl, 6-methoxy derivative (substance 12) was synthesized; however, there was marked loss of chloride sensitivity with preservation of sensitivity to other halides. Methyl substitution at positions 4 and 7 conferred the highest relative intensi-

148

KRAPF

ties (1.63 and 1.30, respectively); 6-methylation conferred the highest molar extinction coefficient (10,500 M-’ cm-‘). The influence of the chemical nature of sub stituents on the quinoline ring was next studied. Tables 2 and 3 show that the compounds with electrondonating substituents (methoxy, methyl: compounds 9, 11, 15-19) had much improved chloride sensitivity compared with those containing electron-withdrawing substituents (chloro, bromo, benzyl: compounds 7,8, 13, 14), with an intermediate chloride sensitivity for the unsubstituted compound (compound 1). In addition, compounds with an electron-donating group have lower peak absorbance wavelengths, fluorescence excitation, and emission wavelengths and shorter fluorescence lifetimes than compounds with electron-withdrawing groups.

TABLE STERN-V• Compound

Cl

ET AL.

The fluorescence of each compound tested was insensitive to sodium, potassium, calcium, phosphate, nitrate, and pH in the range 5-8. The fluorescence intensity of each compound was also insensitive to solution ionic strength above 100 mM; however, a small ionic strength effect was observed as measured by differences between fluorescence lifetimes determined in Na2S04 buffer and distilled water. For each compound, quenching by chloride occurred by a collisional mechanism as judged by parallel decreases in fluorescence intensity and lifetime following addition of 10 mM chloride. It is interesting that the fluorescence of some compounds (compounds 1, 2, 11, 15, and 16) had a significant sensitivity to bicarbonate (but not to pH). The interpretation of this finding in terms of structure-activity rela-

3

LMERCONSTANTS"

Br

I

SCN

HCQ

Citrate

so4

PO4

9 I2 0 0 0 0 0 0

22 27 25 20

0 I 0 0 0 0 0 0

0 0 0 0 0 0 0 0

Ring structure 1 2 3 4 5 6 7 8

55 59 36 26 5 25 3 3

13 81 124 123 224 87 80 68

96 Ill 163 179 307 120 106 110

76 86 128 1’7 255 98 88 90

Ring substituents 9 10 11 12 13 14

118 78 83 26 3 11

175 154 123 134 75 I5

276 233 171 180 109 26

211 186 140 152 84 19

73 87 76 59 97 90

102 118 149 177 148 148

141 156 199 204 213 224

II2 125 161 183 159 170

Ring position 15 16 17 18 19 20 ’ Stern-Volmer

constants

are given

in M-l

5 0 1

29 9 27 19 28 23

STRUCTURE-ACTIVITY

X0

350

400 Wavelength

OF

450

300

500

(nm)

1 o-

0 300

CHLORIDE-SENSITIVE

1 =I

350

350

4CO Wavelength

pm)

400 Waxlength

(nm)

FIG. 2. Fluorescence excitation ofcompounds 7, IO, and 16 at 0. distilled water.

450

500

4.50

500

and emission spectra concentrations in

FLUORESCENT

149

INDICATORS

as citrate. Compounds with the quinoline backbone have much improved chloride sensitivity over those with isoquinoline backbone or those with a 3-ringed backbone. The length of the sulfoalkyl chain is relatively unimportant as is the position of a substituent on the quinoline backbone. Methyl or methoxy group substitution generally increases halide sensitivity: the enhanced halide sensitivity conferred by methyl or methoxy substitution is relatively independent of the ring position substituted, although, quantitatively, substitution of a methoxy group on position 6 yields the highest chloride sensitivity. with loss of bicarbonate sensitivity and a decrease in citrate sensitivity. The bicarbonate sensitivity is preserved when methyl groups are substituted. Substitution of the quinoline backbone with groups having electron-donating properties yields much better chloride sensitivity than substitution with groups having electron-withdrawing properties. These studies establish a set of general guidelines for further synthesis of tailored chloride-sensitive fluorophores. It is likely to be possible to add acetoxymethyl groups to different ring positions without compromising chloride sensitivity. These alterations would yield a molecule that is more diffusible through biological membranes. Within the cell, cytoplasmic esterases would cleave the ester bonds producing a negatively

I-mM

8 tionships, and the question of whether bicarbonate sensitivity can be enhanced by other structural modifications, requires further investigation. Based on the correlations between molecular structure, optical properties, and anion quenching characteristics several conclusions can be drawn. The quaternization of quinoline with a sulfoalkyl group yields a compound with marked halide sensitivity and slight sensitivities to organic anions such

0 0

20

40

Chloride

60

(mMj

FIG. 3. Stern-Volmer plots for quenching of compounds 3, 8, 9, and 11 by chloride. Dye concentration was 0.1 mM in 100 II?M Na2S04. 5 mM Na phosphate. pH 7.4. Data were fitted to Eq. [I] and results arc given in Table 3.

150

KRAPF

charged and relatively impermeable molecule. In addition, via an aliphatic spacer chain attached to the quinoline ring, the addition of a second, chloride-insensitive chromophore (e.g., dansyl) should be possible, providing a method to measure absolute chloride concentration from spectral shape. These studies also provide the necessary basis for synthesis of other types of chloride-sensitive indicators for specialized applications, such as dextran-bound impermeant indicators for cell injection studies or chloride sensitive indicators bound to glass fibers for fiberoptic measurements of chloride concentration. ACKNOWLEDGMENTS The authors acknowledge the advice of Professor Roger Ketchum of the Department of Pharmaceutical Chemistry at USCF. This work was supported by NIH Grants OK35 124 and OK39354, a grant from the National Cystic Fibrosis Foundation, and a grant-in-aid from the American Heart Association with partial support from the Long Beach AHA Chapter. R.K. is the

ET AL. recipient of a generous grant from the Swiss Foundation for medical-biological grants. N.P.I. was supported by NIH Training Grant AM072 19. A.S.V. is an established investigator of the American Health Association,

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iinal. Chem. 314, 577-58

E. (1983) Fresenius’ %. 1. A. S. ( 1987) Biochemis-

2. Illsley, N. P.. and Verkman. tr~,26, 1215-1219. 3. Chen. P.-Y.. Illsley. N. P., and Verkman. A. S. ( 1988) Amer. J. Phwiol.. in press. 4. Chen, P.-Y., and Verkman, A. S. (1988) Biochrmi,stry, in press. 5. Glaubensklee. C.. Illsley. N. P.. Davis, B., and Verkman, A. S. (1987) Biophw J. 51, 344a. 6. Fong, P., Illsley. N. P.. Widdicomhe, J. H.. and Verkman, A. S. (1987) BiophJ!c J. 51, 344a. 7. Krapf. R., Berry, C. A., and Verkman, A. S. (1988) Bi0phy.s. J.. in press. 8. Spencer, R. D.. and Weber. G. (1969) .4nn. N. F. Acad. Pt. 158, 36 l-376. 9. Weber, G. (1977) J. Chem. Ph,w. 66, 4081-409 1, 10. Lakowicz, J. R.. Cherek, H.. and Balter, A. ( 198 I ) J

Biochem. i3iophy.s. Methods 5, 13 I - 146.